In vivo fluorescence and bioluminescence optical imaging are playing an increasing important role in studies of gene and protein expression, and other cellular level activities with very high measurement sensitivity. But their applications are limited by low spatial resolution for deep targets. X-ray imaging is often used as an anatomical imaging tool because of its high resolution. But its application in molecular imaging is limited because of its low contrast sensitivity. We propose a hybrid imaging modality, X-ray luminescence optical tomography (XLOT), to image X-ray excitable nanoparticles in turbid media with both high molecular contrast sensitivity and good spatial resolution, independent of the target depth. A high resolution X-ray beam is used to excite phosphor nanoparticles such as Gd2O2S:Eu (GOS) along a single line through the object. Optical photons emitted by the excited GOS are detected with a sensitive electron-multiplying charge coupled device (EMCCD). By scanning the x-ray beam across the subject, the EMCCD measurements are used to reconstruct the particle concentration using the known excitation locations with a model based reconstruction method. Structural x-ray images also can be produced at the same time. We have built a XLOT prototype system with 2 mm diameter collimated X-ray beam and an EMCCD camera. The X-ray beam scanned a transverse section of phantoms. Embedded cylindrical targets (4.8 mm diameter, 5.7 mm deep) with microscale GOS particles at concentrations of 1.0 mg/mL and 0.2 mg/mL were reconstructed successfully with XLOT but X-ray imaging alone could not detect them due to its low contrast sensitivity. The goal of this proposal is to develop and characterize a XLOT system suitable for in vivo imaging studies, that also can concurrently produce anatomic X-ray CT images. A second goal is to optimize platform phosphor nanoparticle cores that can be functionalized and used with XLOT imaging in support of a broad range of research and development in the rapidly expanding field of nanomedicine.